This invention relates to a pretreatment method for accurately and efficiently discriminating and quantitating cholesterol, which exists in the specific lipoprotein fraction, by simple procedures while using a small amount of a sample, and also to a method for measuring cholesterol in the specific lipoprotein fraction by using the pretreatment method.
Lipids such as cholesterol are complexed with apoproteins in blood to form lipoproteins. Depending on differences in physical properties, lipoproteins are classified into chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and so on. Among these lipoproteins, LDL is known to be one of causative substances which induce arteriosclerosis, while HDL is known to show anti-arteriosclerotic activity.
Epidemiologically, the level of cholesterol in LDL is known to exhibit a positive correlation with the frequency of onset of arteriosclerotic disease while the level of cholesterol in HDL is known to show an inverse correlation with the frequency of onset of arteriosclerotic disease. These days, measurements of cholesterol in LDL or HDL are, therefore, widely conducted for the prevention or diagnosis of ischemic heart diseases.
As methods known for the measurement of cholesterol in LDL or HDL, there are, for example, a method in which LDL or HDL is separated from other lipoproteins by ultracentrifugal separation and is then subjected to a cholesterol measurement; and another method in which subsequent to separation of LDL or HDL from other lipoproteins by electrophoresis, its lipid is stained, and the intensity of a developed color is measured. These methods are however not used practically, because they involve one or more problems in that procedures are intricate and many samples cannot be handled.
A method for the measurement of cholesterol in HDL, which is used at present in the field of clinical tests, is the precipitation method in which a precipitation reagent is added to a sample to agglutinate lipoproteins other than HDL, the resulting agglutinate is removed by centrifugation, and cholesterol in isolated supernatant which contains only HDL is then measured. This method is simpler compared with ultracentrifugation or electrophoresis, but due to the inclusion of the procedures to add the precipitation reagent and to perform the separation, requires each sample is a relatively large quantity, and involves a potential problem of causing an analytical error. Furthermore, the entire analysis steps of this method can not be fully automated.
On the other hand, enzymatic methods have been studied for the fractional quantitation of cholesterol in HDL. Known methods include, for example, to conduct an enzymatic reaction in the presence of a bile acid salt and a nonionic surfactant (JP 63-126498 A). This method makes use of the fact that an enzymatic reaction proceeds in proportion to the concentration of cholesterol in LDL in an initial stage of the reaction and the subsequent reaction velocity is in proportion to the concentration of cholesterol in HDL. A problem however exists in accuracy because the reaction with the cholesterol in HDL and the reaction with cholesterol in other lipoproteins cannot be fully distinguished.
Also included in the known methods is to have lipoproteins other than HDL agglutinated in advance, to cause cholesterol in HDL alone to react enzymatically, and to inactivate the enzyme and at the same time, to redissolve the agglutinate, followed by the measurement of an absorbance (JP 6-242110 A). This method, however, requires at least three procedures to add reagents so that it can be applied only to particular automated analyzers, leading to a problem in a wide applicability. Further, this method is not satisfactory from the standpoint of damages to analytical equipment and disposal of the reagents because of the use of a salt at a high concentration upon redissolution of an agglutinate.
A still further method is also known (JP 9-299 A), which comprises causing, in a first reaction, cholesterol oxidase and cholesterol esterase to act upon lipoproteins other than HDL in the presence of a special surfactant and to have cholesterol, which is contained in such other lipoproteins, preferentially reacted, and then measuring cholesterol in HDL while inhibiting any reaction to cholesterol in lipoproteins other than HDL. This method, however, is considerably different from the present invention inter alia in that in the first reaction, the special surfactant, cholesterol oxidase and cholesterol esterase are required at the same time to put, outside the reaction system, both free cholesterol and esterified cholesterol in the lipoproteins other than HDL.
Further, Japanese Patent No.2,600,065 discloses a method which makes combined use of a precipitation reagent, which is adapted to cause precipitation of lipoproteins other than HDL, and a cholesterol measuring reagent to measure cholesterol (HDL-C) in unprecipitated HDL. This method has practical utility when a modified enzyme is used as enzyme and xcex1-cyclodextrin sulfate is used as a precipitation reagent. This method, however, also involves a problem in accuracy in that turbidity, which occurs as a result of the use of the precipitation reagent, interferes with the measurement system.
Concerning the measurement of HDL-C by a modified enzyme, xe2x80x9cSEIBUTSU SHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)xe2x80x9d, 19(5), 305-320, which is considered to be a published paper on the above-described patented method, discloses that, under the recognition of incapability of measurement of HDL-C in a serum of a hyperlipidemic patient by the modified enzyme due to a positive error (that is, to result in a higher value compared with that obtained by the precipitation method) induced when the modified enzyme is simply introduced into a reaction system, HDL-C was measured by using cyclodextrin sulfate, a polyanion, and magnesium chloride as a precipitation reagent for the avoidance of the positive error.
To reduce the influence of turbidity caused by a precipitation reagent in the above-described patented method, certain techniques are also known, including to make a surfactant exist concurrently (JP 8-116996 A), to use an antibody (JP 9-96637 A), and to employ a sugar compound (JP 7-301636 A). They, however, all require as a premise the inclusion of a reagent which induces formation of an agglutinate, so that it is fundamentally indispensable for them to use a precipitation reagent such as a polyanion.
The present inventors recently found that use of a substance, which acts upon the specific lipoprotein only, makes it possible to accurately quantitate cholesterol in the specific lipoprotein fraction without using a precipitation reagent, and filed patent applications (JP 9-244821). This method has an extremely high correlation with the conventional precipitation method, but compared in measurement values with the precipitation method, this method is recognized to have a similar tendency as the above-described method reported in xe2x80x9cSEIBUTSU SHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)xe2x80x9d. To obtain data consistent with those obtained by the conventional precipitation method at medical institutions and the like, a polyanion or the like is added.
From the standpoint of the problem of a tarnish or the like on a cuvette and scattering of measurement values, however, it is not desired to add a polyanion or the like and to form a precipitate in a measurement system. Accordingly, it has been strongly desired to eliminate the precipitate from the system. Further, it is also economically unreasonable to use a polyanion or the like for making the resulting data consistent with those obtained by the precipitation method although the polyanion or the like is not needed from the standpoint of the principle of the measurement. Hence, there is also an outstanding desire for its solution.
An object of the present invention is, therefore, to provide a method, which can accurately and efficiently quantitate cholesterol in the specific lipoprotein fraction by simple procedures fundamentally without needing a polyanion or the like and is suitably applicable to various automated analyzers.
The present inventors proceeded with a thorough investigation for a cause which may be responsible for the above-described problem reported in xe2x80x9cSEIBUTSU SHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)xe2x80x9d, that is, the problem that a value of cholesterol in the specific lipoprotein fraction as quantitated by using a substance which acts only upon a specific lipoprotein such as HDL becomes higher than the corresponding value as determined by the precipitation method; and came to a conclusion that even from non-HDL lipoproteins (LDL, VLDL and the like) the cholesterol of which is not supposed to be measured, a small amount of free cholesterol existing on their surfaces or in the vicinity of their surfaces is liberated to cause a positive error. Based on this finding, it has been found that a cholesterol value obtained by a quantitation method making use of a substance, which acts upon a specific lipoprotein only, becomes consistent with the corresponding value obtained by the precipitation method when the cholesterol value is measured after consuming only free cholesterol in advance under conditions that lipoproteins remain substantially unchanged, leading to the completion of the present invention.
Described specifically, the present invention provides a method for pretreating a sample, which contains various lipoproteins, prior to measuring cholesterol existing in specific one of the lipoproteins in the sample, which comprises causing an enzyme, which acts upon free cholesterol as a substrate, to act upon the sample to consume only the free cholesterol in advance under conditions that the lipoproteins remain substantially unchanged.
The present invention also provides a method for quantitating cholesterol existing in a specific lipoprotein in a sample, which comprises causing an enzyme, which acts upon free cholesterol as a substrate, to act upon the sample with the lipoprotein contained therein to consume only the free cholesterol under conditions that the lipoproteins remain substantially unchanged; and then measuring the cholesterol, which exists in the specific lipoprotein, by using a substance which acts upon the specific lipoprotein only.